Strategy for Sensitive and Specific Detection of Yersinia pestis in Skeletons of the Black Death Pandemic

Abstract

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14th century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

United in death—related by blood? Genetic and archeometric analyses of skeletal remains from the neolithic earthwork bruchsal‐aue

“…Possible causes of death for multiple burials have been previously discussed and sometimes proven, e.g., in the case of the violent extermination of 13 individuals interred in the four multiple burials of the Corded Ware Culture graveyard of Naumburg‐Eulau in Central Germany (Haak et al, ), indicated by multiple traumatic injuries. When the context of a multiple burial allows such concrete hypotheses, even infectious diseases can be detected with paleogenetic methods (Seifert et al, , Harbeck et al, ).…”

“…For prevention of contamination, a catalogue of precautional measures was applied, including physically separated pre‐ and post‐PCR workspaces. Sample preparation, DNA extraction and PCR‐setup of ancient samples were performed in the clean laboratory of the ArchaeoBioCenter of the Ludwig‐Maximilians‐University Munich as described by Wiechmann et al () and Seifert et al (). Modern samples, positive controls and the oligonucleotide for quantification were exclusively handled in the clean laboratory at the Faculty of Biology, Ludwig‐Maximilians‐University Munich, where qPCRs, PCRs and sequencing were performed.…”

Early medieval stone‐lined graves in Southern Germany: analysis of an emerging noble class

“…Bold numbers 5 graves investigated; highlighted in grey 5 stone-lined graves; black line 5 5 meters scale. Sexing was conducted examining seven pelvic features and the index ischio-pubicus (Novotn y, 1972), as well as eight features of the skull and mandible (Ferembach, Schwidetzky, & Stloukal, 1979;R€ osing et al, 2005;Steckel, Larsen, Sciulli, & Walker, 2005 Sample preparation, aDNA extraction and analyses of mtDNA and Y-STRs were carried out as previously described (Keller et al, 2015;Seifert et al, 2013). Following Martin (1928), age at death was classified into the following stages: Infans I (0-6 years), infans II (7-12 years), juvenile (13-20 years), adult (21-40 years), mature (41-60 years), senile (60-x years).…”

“…Bold numbers 5 graves investigated; highlighted in grey 5 stone-lined graves; black line 5 5 meters scale. The plans are altered according to Keller (1995) Sample preparation, aDNA extraction and analyses of mtDNA and Y-STRs were carried out as previously described (Keller et al, 2015;Seifert et al, 2013). Quantification of mitochondrial molecules per reaction was achieved using a 100 bp fragment covering a diagnostic SNP at position 7028 in the mitochondrial genome which can be used to distinguish between H-and non-H haplogroups (Melchior, Gilbert, Kivisild, Lynnerup, & Dissing, 2008).…”

Pest und Lungenpest

“…Erst in den letzten Jahren gelang der bestätigte Nachweis von Y.-pestis-DNA aus historischen Skeletten. In den Zähnen der Opfer konnte der Pesterreger mittels molekularbiologischer Methoden identifiziert, sequenziert und sogar typisiert werden [5,6].…”